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SURVEY OF WHITEFLIES AND THEIR TRANSMISSIONOF PLANT VIRUSES IN TAIWAN

Chiun-Cheng Ko1, Shin-Chung Chang2, and Chung-Chi Hu3

1Department of Entomology, National Taiwan University (NTU), Taipei, Taiwan; 2Bureau of Animal and Plant Health Inspection and Quarantine (BAPHIQ),

Council of Agriculture (COA), Taipei, Taiwan; and3Graduate Institute of Biotechnology, National Chung Hsing University,

Taichung, Taiwan

ABSTRACT

On a global basis, there are more than 1,420 known species of whiteflies (Hemiptera:Aleyrodidae). Extensive collections of whitefly species have been made in Taiwan. So far, thenumber of species has increased to 158 species in 45 genera. Among whitefly species, Bemisiatabaci is polyphagous and exhibits morphological polymorphism on different host plants, withmore than 24 biotypes having been discriminated by specific phytotoxic reactions, esterasemarkers, and DNA markers. Thus, the objective of this study was to examine the taxonomic statusof B. tabaci populations in Taiwan by molecular markers. First, molecular markers of mtCOI, 16S,and ribosomal ITS1 were used to reconstruct the phylogeny of B. tabaci populations in Taiwan.The results indicated that there are three biotypes (B, An, and Nauru) in Taiwan. Second, wedeveloped sequence-characterized amplified region (SCAR) markers for each of six B. tabacibiotypes (A, B, Q, Nauru, AN, and S). Six of these SCARs were useful in distinguishing eachbiotype from the others. Finally, we proceeded to collect and characterize the begomoviruses inseveral weeds commonly seen in the fields of Taiwan, and demonstrated that the begomovirus fromGonostegia pentanda may be transmitted to tomato plants, and vice versa, by whiteflies. Theresults may provide valuable insights into the understanding of the life cycles of begomovirusesand the design of effective disease management methods for them in Taiwan.

Key words: Aleyrodidae, Bemisia tabaci, molecular markers, begomovirus, Taiwan

INTRODUCTION

Whiteflies, together with aphids, scale insects,and psyllids, belong to a family of tiny insectscalled Hemiptera (Sternorrhyncha). The lifecycle of whiteflies has three stages, namely,the egg, the first to fourth instar nymphs, andthe adult. All identifications are based on thefourth instar nymph (popularly referred to asthe pupal case). On a global basis, whiteflieshave more than 1,420 known species, amongwhich 1,000 species have been described fromthe tropics but only 420 from temperate areas.Among the 151 recorded genera, over halfhave records of only one or two species, andonly eight genera exceed 50 species. However,whiteflies have been studied mainly by workersin temperate areas, and in the tropics, our

knowledge is mostly limited to that from a fewlocal specialists (Fig. 1). Research intowhiteflies has been relatively ignored in tropicalareas. We believe that the number of whiteflyspecies will increase if more detailed studieswere conducted. Much remains to be learnedfrom exploring the whitefly fauna of the world.

The taxonomy of whiteflies is mainlybased on pupal (fourth instar skin) characters,but this is complicated by the fact thatintraspecific morphological variabilities in thepuparium, namely, the number, size, shape, andposition of setae and of papillae, the perianalstructure, and the body size, are quite commonin response to environmental variables,particularly, the degree of hirsuteness of thehost plant. For example, Bemisia tabaci(Gennadius) has several junior synonyms, and

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is a globally serious pest which possessesmore than 24 biotypes; these biotypes aredifficult to distinguish by morphologicalcharacters. Studies suggest that B. tabacishould be considered a species complex(Perring 2001; Ko et al. 2002). In this regard,supplemental information for developingmolecular and biochemical techniques is neededfor determining with added confidence whichtaxa are true genera and species and which areillusory.

The role of whiteflies in agriculturalecosystems is becoming increasingly important.The main reason is the frequent trade ofinternational agricultural products, and themigration and introduction of new pests due tohuman activities (Tables 1, 2). Excessive B.tabaci-induced losses have occurred worldwidein vegetable and ornamental crop production.Losses occur from plant diseases caused by B.tabaci-transmitted viruses, direct feedingdamage, plant physiological disorders, and

Fig. 1. Whitefly taxonomists in Asian countries. Numbers indicate species numbers.(C, Corbett; C&Y, Chou and Young; D, David; J, Jesudasan; K, Ko; Ku, Kuwana; M,Meganathan; P, Peal; Q&B, Quaintance and Baker; R, Regu; S, Singh; Su, Sundararaj;T, Takahashi; Y, Young)

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honeydew contamination and associated fungalgrowth. The number of B. tabaci-transmittedplant viruses has increased, and total yieldlosses of important food and industrial cropshave likewise increased. Begomoviruses haveemerged as constraints to the cultivation of avariety of crops in various parts of the world.Some of the diseases caused bybegomoviruses are devastating. The frequencywith which new begomoviruses are appearingshows that these viruses are still evolving andpose a serious threat to sustainable agriculture,particularly in the tropics and subtropics. Inaddition, with adaptations by whiteflies and theincreased international trade of plant materials,whitefly-transmitted viruses are expected to beamong major plant pathogens in Taiwan in thefuture.

MOLECULAR IDENTIFICATION OFWHITEFLY SPECIES IN TAIWAN

Biotypes have been identified in different areasof the world suggesting that B. tabaci may be

a species complex undergoing evolutionarychange. These biotypes may exhibit differencesin viruses transmitted and transmissionefficiencies, rates of development,endosymbionts, host utilization, andphysiological host damage (Oliveira et al. 2001).

Distribution and Identityof Bemisia tabaci in Taiwan

We collected 202 samples covering a wide areaof Taiwan. The molecular markers of mtCOI,16S, and ribosomal ITS1 were used toreconstruct the phylogeny of B. tabacipopulations in Taiwan. The results indicatedthree biotypes (B, An, and Nauru biotypes),including 129 collections of the B biotype, 62collections of the Nauru biotype, and 11collections of the An biotype in Taiwan(Tables 3, 4) (Figs. 2, 3) (Hsien et al.unpublished data).

Table 1. Whiteflies introduced in Taiwan

Whitefly species Approximate year of discovery in Taiwan

Pealius spina (Singh 1931) ?Pealius azaleae (Baker & Moles 1920) ?Pealius rhododendri (Takahashi 1935) 1930sAleyrodes proletella (Linnaeus 1758) ?Trialeurodes vaporariorum (Westwood 1856) 1988Aleurodicus dispersus (Russell 1965) 1988Bemisia argentifolii (Bellows & Perring 1994) 1990?Paraleyrodes bondari (Peracchi 1971) 1998

Table 2. The most-damaging whitefly pests recorded in Taiwan

Whitefly species Host plants recorded Host plants recordedin Taiwan worldwide

Trialeurodes vaporariorum > 50 species 281 species in 82 familiesAleurodicus dispersus 156 species in 65 families > 250 speciesBemisia argentifolii 169 species in 38 families > 600 speciesParaleyrodes bondari 25 species in 17 families ?

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Sequence-characterized AmplifiedRegion (SCAR) Markers for Biotypesof Bemisia tabaci

Molecular detection methods using randomamplified polymorphic DNA (RAPD) productswere developed to distinguish the biotypes.This method is convenient because samplescan be preserved in alcohol, and relativelylarge numbers of samples can rapidly beprocessed; however, the banding patterns arecomplex, and it takes experience to correctlyinterpret them. Sequence-characterized amplifiedregions (SCARs) can be developed to amplifyonly one product, and this makes the resultseasier to interpret.

Total DNA of B. tabaci was isolated fromindividual whiteflies which had been pulverizedin a pestle in 25 µl lysis buffer (50 mM KCl,10 mM Tris (pH 8.4), 0.45% Tween 20, 0.2%gelatin, 0.45% NP40, and 60 µg/ml proteinaseK) and incubated at 65°C for 30 min (De Barroand Driver 1997). These DNA samples wereanalyzed using 86 10-mer Operon primers(Operon Technologies, USA). The OPA-15primer (5’ CACCAGAAGT 3’) generated a bandof 1100 bp which was present in the Abiotype but absent from the other biotypestested. This fragment was cloned into pGEM-T (Promega, USA), its extremes sequenced, and2 sets (BaAF/BaAR) of approximately 20-meroligonucleotide primers were designed. On theother hand, the BaQF/BaQR primer set wasdesigned based on the OPO-18 primer (5’GTCATCCAGA 3’) which generated a band of1100 bp present in the Q biotype. Four forward primers were designed for theother four biotypes from comparisons ofaligned mitochondrial gene COI sequencesamong biotypes. Each of these primers wasable to match with the COI reverse primer, L2-N-3014, to discriminate the different biotypes.

These SCAR markers were tested usingdifferent biotypes and 12 different whiteflyspecies. PCR reactions were carried out in atotal volume of 25 µl with 5 µl of a template(DNA extracted as above), 0.15 µM dNTPs, 2.5mM MgCl2, 1 unit Taq DNA polymerase, 0.6µM of each primer, and 2.5 µl of 10x Taqreaction buffer. The cycling protocol was asfollows: 1 cycle at 94°C for 5 min followed by35 cycles at 94°C for 1 min, 56-66°C for 1 min,

and 72°C for 1 min. Amplified products wereseparated on 1.5% agarose gels.

The six SCAR markers we developed weresuccessfully used to discriminate each biotypefrom the other six biotypes and 12 differentwhitefly species. All of these markers yieldedidentification PCR products in each biotype.Although BaQF/BaQR was unable to amplify asingle band from biotype Q, it could stilldiscriminate biotype Q from the other biotypesand different whitefly species (Fig. 4). Thesimplicity of the amplified products means thatthe results are easy to interpret. In order toassure that these primers are robust, samplesfrom many different countries need to betested (Hung et al. unpublished data).

WHITEFLIES AND VIRUS TRANSMISSIONIN TAIWAN

Modes of transmissionof viruses by whiteflies

The plant viruses known to be transmitted bywhiteflies in Taiwan and in the world are listedin Tables 5 and 6, respectively. The modes oftransmission of plant viruses are characterizedinto at least three types, persistent, semi-persistent, and nonpersistent, mainly based onthe time required for the vector to acquire theability to transmit the virus (acquisition time),and the length of time the vector retains thatability (retention time). The choices ofstrategies for virus and vector control dependheavily on the modes of transmission of theviruses by the vectors.

Ipomoviruses are transmitted by whitefliesin a nonpersistent manner (Hollings et al. 1976,Liao et al. 1979), with acquisition and retentiontimes of a few seconds and several minutes,respectively. Thus, whiteflies might acquire andtransmit the viruses before they are killed byinsecticides, which would render the usualstrategy of chemical control ineffective.Methods such as silver plastic covers andshiny strips that prevent the invasion of, orrepel, whiteflies, might be useful alternatives.For criniviruses, carlaviruses, andclosteroviruses, whiteflies transmit pathogens ina semi-persistent mode, acquiring the viruses ina few minutes and retaining the viruses for afew hours or days until they molt (Schaefersand Terry 1976; Duffus et al. 1986; Horn et al.

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1989). Usual insecticidal controls may thus beapplied for the management of such viruses.

Relationships between whiteflies andbegomoviruses, which have emerged as seriouscrop threats causing devastating diseases ofeconomically important crops (Moffat 1999), arethe most extensively studied among whitefly-borne viruses, and are the focus of thissection of the report. For begomoviruses, themode of transmission by whiteflies iscategorized as persistent (Goodman and Bird1978), with a long acquisition period (15 min toa few hours), an additional 6-12 hr of a latencyperiod before a transmission event, and acontinual viral retention time throughout theirlifespan. The time required for successfultransmission is quite long, ranging from a fewhours to days (Bird and Maramorosch 1978;Cohen et al. 1989). The begomoviruses alsocirculate in the body fluids of whiteflies.There is evidence suggesting the transovarial

passage of begomoviruses to progeny (Ghanimet al. 1998) and the lateral transmission amongthe adult whiteflies in a sex-related manner(Ghanim and Czosnek 2000). The persistence ofa begomovirus transmission capability and theaccumulation of viral DNAs inside whitefliessuggest that begomoviruses may replicate to acertain level in whiteflies. Therefore, theeffective control of whiteflies by insecticides isof vital importance for the disease managementof begomoviruses.

Common weeds in the fields asalternative or overwinteringhosts for begomoviruses

In the early 1990s, geminiviruses caused up to95% yield losses of tomatoes in the DominicanRepublic (Moffat 1999), and inflicted over US$2billion/year yield losses in cassava in Africa(Harrison and Robinson 1999). In Brazil,

Table 3. Biotype, sample location, host plant and GeneBank accession number forBemisia tabaci mtCOI sequences (Hsien et al. unpublished data)

Acronym Biotype Geographical Host plant GeneBanklocation/year Accession no.

TWNauru1 Nauru Hsinchu, Taiwan/2003 Boehmeria nivea DQ174518TWNauru3 Nauru Chiay, Taiwan/2002 Humulus scandens DQ174519TWNauru4 Nauru Taidung, Taiwan/2003 Ipomoea acuminate DQ174520TWNauru10 Nauru Kaohsiung, Taiwan/2002 Euphorbia pulcherrima DQ174521ChinaNauru16 Nauru Jiangsu, China/2004 Gossypium hirsutum DQ174522ChinaNauru17 Nauru Guangdong, China/2004 Codiaeum variegatum DQ174523IndonesiaF16 Nauru Indonesia/2003 Capsicam annunm DQ174524TWAn1 AN Chiayi, Taiwan/2002 Momordica ochinchinensis DQ174525TWAn2 AN Kaohsiung, Taiwan/2003 Achyranthes obtusifolia DQ174526TWAn3 AN Hualien, Taiwan/2003 Mesona chinensis DQ174527TWAn4 AN Hsinchu, Taiwan/2003 Solanaceae DQ174528AusAnF3 AN Australia - DQ174529TWB1 B Taidung, Taiwan/2003 Fabaceae DQ174530TWB2 B Taichung, Taiwan/2004 Euphorbia pulcherrima DQ174531TWB3 B Kaohsiung, Taiwan/2003 Lycopersicon esculeutum DQ174532TWB4 B Tainan, Taiwan/2003 Lycopersicon esculeutum DQ174533TWB5 B Matsu, Taiwan/2003 Euphorbia pulcherrima DQ174534AusBF5 B Australia - DQ174535IsraelBF17 B Israel/1997 Hibiscus rosa-sinensis DQ174536JapanBF18 B Hirashima, Japan/1998 Brassica oleracea DQ174537KoreaBF22 B Goyang, Korea/2003 Rosa hybride DQ174538SpainQF7 Q Spain/2002 - DQ174539SpainQF12 Q Spain/2002 - DQ174540NetherlandsQF13 Q the Netherlands/2002 Hibiscus sp. DQ174541USAAF1 A USA/2002 - DQ174542

Table 4. Host plants of Bemisia tabaci in Taiwan (Hsien et al. unpublished data)

Host plant Bemisia tabaci biotypeFamily Species B Nauru An

Acanthaceae Dicliptera chinensis +Amaranthaceae Achyranthes obtusifolia + +

Achyranthes bidentata + + +Achyranthes rubrofusca + +

Asteraceae Ageratum conyzoides +Ageratum houstonianum +Bidens chilensis +Bidens pilosa +Chromolaena odorata +Eclipta prostrata +Emilia sonchifolia + + +Erigeron bonariensis +Gaillardia aristata +Ixeris chinensis +Lactuca sativa +Siegesbeckia oriental + +Sonchus oleraceus + + +Wedelia trilobata +Xanthium strumarium +Youngia japonica +

Brassicaceae Brassica oleraceae +Brassica rapa +

Convolvulaceae Ipomoea acuminata +Ipomoea batatas +Ipomoea obscura +

Cucurbitaceae Cucumis anguria +Cucumis melo +Cucurbita moschata + +Luffa aegyptiaca +Momordica ochinchinensis +Sechium edule +

Euphorbiaceae Euphorbia cyathophora +Euphorbia hirta + +Euphorbia pulcherrima + +Jatropha podagrica +

Fabaceae Leucaena glauca +Lamiaceae Mentha arvensis +

Mesona chinensis +Leonurus heterophyllus + +

Malvaceae Hibiscus sabdariffa +Sida cordifolia +Sida sp. + +Urena lobata + +

Moraceae Humulus scandens + +Myricaceae Myrica rubra +Portulacaceae Portulaca oleracea +Solanaceae Lycopersicon esculeutum + +

Nicotiana tabacum +Physalis angulata +Solanum nigrum +

Urticaceae Boehmeria nivea +

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A biotype

cassavabiotype

Ms biotype

Q biotype

B biotype

Naurubiotype

An biotype

0.05 substitutions/site

Seychelles Ms

MadagascarMs

MauritiusMsReunionMs

NetherlandsQF13Turkey

Ivory CoastO

SpainQF7Spain1Q

SpainQF12

MoroccoQAusBF5

IsraelBF17TWBH1

TWBH4

FranceBTWBH3

SouthAfricaB

ArizonaB

KoreaBF22TWBH2

ChinaBJapanBF18TWBH5

India1TWNauruH10

ChinaNauruH17

IndonesiaF16ChinaNauruH16

NepalTWNauruH3

TWNauruH1

Pakistan1India2

TWNauruH4

AusANF3TWAN4

TWAN3

China1TWAN1

TWAN2

PakistanThailand1

Malaysia

UgandaBCUgandaBNaC

UgandaBIC

UgandaBuCUgandaBMC

Ghana

Malawi

Tanzania

ColombiaA

USAAF1MexicoA

ArisonaA

Bolivia

ArgentinaBemisia afer

100

9876

100

98

100

6064

91

98

74

100

100

100

100

100

95

100

99

8881

88

98

100

6990

10099

100

88

71

86

100

67

Fig. 2. Neighbor joining tree for mtCOI sequences of Bemisia tabaci obtained fromTaiwan, neighboring islands, and GeneBank. [Numbers at note represent thepercentage of 1,000 bootstrap. There are B, An, and Nauru biotypes in Taiwan.The outgroup is Bemisia afer (Hsieh et al. unpublished data).]

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Fig. 3. Distribution of three biotypes in Taiwan and neighboring islands.(A) B Biotype, (B) Nauru biotype, (C) An biotype (Hsien et al.unpublished data).

Fig. 4. Application of molecular identification techniques for Bemisia tabaci biotypes byPCR. (A) BaAF/BaAR primer set for biotype A; (B) BaBF/L2-N-3014 primer setfor biotype B; (C) BaQF/BaQR primer set for biotype Q; (D) BaNaF/L2-N-3014primer set for biotype Nauru; (E) BaANF/L2-N-3014 primer set for biotype AN;(F) BaSF/L2-N-3014 primer set for biotype S (Hung et al. unpublished data).

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Table 5. Names, hosts, and distribution of plant viruses transmissible by Bemisia tabaciin Taiwan

Virus Main natural hosts Distribution References

Ageratum yellow vein Ageratum sp. Taiwan Fauquet et al. 2003,Taiwan virus: AYVTV; Jones 2003Begomovirus

Poinsettia leaf curl virus: Euphorbia pulcherrima Taiwan Tsai et al. 1997,PLCV; Begomovirus Jones 2003

Sweet potato leaf curl Ipomoea batatas Taiwan, USA Lotrakul et al. 1998,virus: SPLCV; Fauquet et al. 2000,Begomovirus Fauquet et al. 2003,

Jones 2003

Tomato leaf curl Taiwan Lycopersicon Taiwan, Japan Fauquet et al. 2000,virus: ToLCTWV; esculentum Fauquet et al. 2003,Begomovirus Jones 2003, Varma

and Malathi 2003

Sweetpotato dwarf virus: Ipomoea batatas Taiwan, Japan Jones 2003, VarmaSPYDV; Ipomovirus and Malathi 2003

Table 6. Categories of whitefly-transmitted plant viruses

Example Species Genus/Family Mode References

Bean golden mosaic virus Begomovirus/ Persistent Goodman and Geminiviridae Bird 1978

Sweetpotato mild mottle virus Ipomovirus/ Non-persistent Hollings et al. Potyviridae 1976; Liao et al.

1979

Lettuce infectious yellow virus Crinivirus/ Semi-persistent Duffus et al. 1986 Closteroviridae

Sweetpotato sunkenvein virus Closterovirus/ N/A* Schaefers and Closteroviridae Terry 1976

Cucumber vein yellowing virus unassigned/ Semi-persistent Sela et al. 1980 Potyviridae

Cowpea mild mottle virus Carlavirus/ Semi-persistent Horn et al. 1989 Flexiviridae

Urd bean leaf crinkle virus unassigned/ N/A Narayanasamy and unassigned Jaganathan 1974

* N/A: No information available.

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economic loss estimates were not made, but inthe last four years, more than 11,000 jobs havebeen lost in the tomato industry because ofwhitefly-transmitted geminiviruses and otherfactors. In Taiwan, geminiviruses, often inassociation with other viruses, have devastatedtomato fields in the southern and centralregions in recent years, occasionally causingtotal losses in some fields. As whitefliesbecome more adapted to local climates andenvironments, it is foreseeable thatgeminiviruses will become one of the majorconstraints on agricultural production inTaiwan.

Most researches have focused ongeminiviruses that are isolated directly frominfected crop plants. However, weeds alsoserve as important alternative or overwinteringhosts for begomoviruses. The survival ofbegomoviruses in the postharvest season orduring unfavorable conditions and the geneticrecombination of begomoviruses in the hostsor vectors have been the focus of recentepidemiological studies. For instance, Hewittiasublobata (Euphorbiaceae) is widespreadthroughout the tropics and has been found toserves as a host for the East African cassavamosaic virus. It has also been shown thatAgeratum yellow vein virus can infect Frenchbean and tomato, suggesting that Ageratumconyzoides may act as a reservoir host for thepathogen (Tan et al. 1995).

Therefore, we proceeded to collect andcharacterize the begomoviruses on severalweeds commonly seen in fields in Taiwan, anddemonstrated that begomoviruses fromGonostegia pentanda (Huang et al.unpublished data) can be transmitted to tomatoplants, and vice versa, by whiteflies. Theresults may provide valuable insights intounderstanding life cycles of and for designingeffective disease management methods forbegomoviruses in Taiwan.

WHITEFLY TRANSMISSION OFGEMINIVIRUSES FROM GONOSTEGIA

PENTANDRA TO TOMATO (LYCOPERSICONESCULENTUM)

Pre-inoculation Screening

Non-viruliferous B. tabaci were kindly providedby F. C. Lin (Taiwan Agricultural Research

Institute, Taichung, Taiwan), and maintained onNicotiana benthamiana in insect-proof cageswith a 24-hour feeding time. The tomatocultivar used in this study is Known-You 301(give vendor info and location), one of themost commonly cultivated varieties in Taiwan.To ensure the absence of begomovirusesbefore inoculation, all plant materials were keptin whitefly exclusion cages (polyethylene 400-mesh) and kept in a greenhouse understandard conditions. The whiteflies and plantmaterials were also checked for begomovirusinfection by a modified PCR as described byWyatt and Brown (1996).

Whitefly Transmission Tests

Protocols for the whitefly transmission testwere adopted from Brown and Nelson (1988),with modifications. Fifty non-viruliferous B.tabaci were allowed to acquire thebegomoviruses from symptomatic G. pentandrain the whitefly-exclusion cages for 24 hours.The whiteflies were then collected byaspiration, transferred onto the test plants, andallowed to transmit the viruses to fivebegomovirus-free tomato plants maintainedseparately in whitefly-exclusion cages for twodays. The whiteflies were then eliminated byspraying with the insecticide endofulfan. Leafsamples were collected 28 and 42 days post-inoculation and assayed for the presence ofbegomoviruses by PCR. The experiments wererepeated three times.

Detection of Begomoviruses by PCR

The presence of begomoviruses in planttissues was assayed essentially following themethods of Wyatt and Brown (1996), withmodifications. Briefly, 0.2 g of leaf sample wasground in 1 ml of grinding buffer (50 mM Tris-HCl and 10 mM EDTA; pH 8.0). The extracts(50 µl) were transferred to sterile polypropylenetubes, incubated on ice for 30 min, andwashed 3 times with 200 µl of grinding buffer.The DNA bound on the tubes was useddirectly as templates for the PCR detection.Each 20-µl PCR reaction contained 10 pmole ofa primer pair specific to the conservedsequences of the begomovirus coat proteingenes, GCP1 (5’-GGCATTGCCATGGCGAAGCGACCCGCCG-3’) and GCP2 (5’-GCGCGGATCC

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TTAATTTTGTATCGAATCATAG-3’), and 2.5units of rTaq DNA polymerase (Takara) plus10 mM dNTP and 1.5 mM MgCl2.Amplification was carried out in a Gene Amp®PCR System 2400 (Perkin Elmer, PE)programmed to perform 1 cycle of 2 min at94°C, followed by 30 cycles of 1 min at 94°C,1 min at 60°C, and 3 min at 72°C, with a finalextension for 5 min at 72°C. The PCR productswere analyzed by electrophoresis through a 1%agarose gel, followed by cloning andsequencing analysis (Sanger et al. 1977). Thenucleotide sequences were further analyzed forsimilarities and phylogenetic relationships withthose of other begomoviruses in the databaseusing the Gap, PileUp, and PaupSearchprograms of the GCG Package (WisconsinPackage vers. 10.3, Accelrys, San Diego, CA)or the ClustalW program (Higgins et al. 1992).

Results and Discussion

Mixtures of diverse begomoviruses wereassociated with both Bemisia tabaci and G.pentandra. To investigate the relationshipbetween whiteflies and G. pentandra, the coatprotein (CP) genes of begomoviruses fromwhiteflies collected from symptomatic tomatoesin fields in Nantou County, central Taiwan, andsymptomatic G. pentandra plants wereamplified (Figs. 5, 6), cloned, and molecularlycharacterized, as the CP gene plays a key rolein determining virus-vector and virus-hostspecificities (Harrison and Robinson 1999). Intotal, 58 independent clones were sequenced,and those with sufficiently distinct sequences(<95% sequence identity) were used in thephylogenetic analyses. As shown in Figs. 7and 8, mixtures of diverse begomoviruses were

Fig. 5. Agarose gel electrophoresis of PCR-amplifiedproducts representing begomoviruses coatprotein genes (CP) from whitefly extracts.Begomovirus CP genes were amplified by PCRusing GCP-1 and GCP-2 primer pair, withwhitefly extracts as templates. The identitiesof the samples are as indicated on the top ofeach lane. The position of the expected CPfragment is indicated by the arrow on theright (Huang et al. unpublished).

Fig. 6. Agarose gel electrophoresis of PCR-amplifiedproducts representing begomoviruses CP genesfrom G. pentandra. Begomovirus CP geneswere amplified by PCR using GCP-1 andGCP-2 primer pair, with G. pentandra grownin Nantou as templates. The identities of thesamples are as indicated on the top of eachlane. The position of the expected CPfragment is indicated by the arrow on theright (Huang et al. unpublished).

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found in both whiteflies and G. pentandraplants. The begomoviruses in B. tabaci werefound to be closely related, sharing >92%sequence identity, to the tomato leaf curl virusTaiwan strain (TLCV-Tw) (Chen et al.unpublished data), whereas most of those fromG. pentandra were distinct from all knownbegomoviruses, with <80% sequence identities.However, at least three clones, gcp5, g3-2, andg1, were found to be more closely related toTLCV-Tw, with >90% sequence identities. Inaddition, patches of sequences with very highsimilarities to TLCV-Tw in both whiteflies andG. pentandra were also revealed (not shown).

Recombination events have been the cause ofmajor outbreaks of new epidemics ofbegomoviruses. For instance, observations andrecords indicate that an epidemic caused by anew cassava-infecting virus in Uganda, UgV,spread from a small area of Uganda to coveralmost the entire country, with cassavagrowing being largely eliminated in the areasaffected. It was found that the nucleotidesequence of UgV DNA-A was essentially thesame as that of the East Africa cassava mosaicvirus, except that the central 60% of the CPgene was virtually the same as that of Africacassava mosaic virus (Zhou et al. 1997).

wcp4

wcp5

wcp7

wcp2

wcp3

tlcv-twcp

wcp1

wcp6

ayw-pdcp

gonocp

g9cp

0.1

Fig. 7. The phylogram reconstructed with the program Clustal W depicting distancerelationships of nucleotide sequences of geminiviruses coat protein fromwhiteflies. The tlcv-twcp, Taiwan strain of TLCV; ayvv-pdcp, Ping-Tung strain ofAYVV; g9cp, gonocp, CP gene sequences of geminiviruses from G. pentandra;wcp1 through wcp6, CP gene sequences of geminiviruses from whitefly extract.Scale bar at the lower left represents the 0.1 unit of phylogenetic distance(0.1 nucleotide substitution per position) (Huang et al. unpublished).

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These results indicated that B. tabaci in thefield may serve as a “natural hotbed” forsequence recombination, since variousbegomoviruses may be acquired by B. tabacifrom different host plants.

Begomoviruses from symptomatic G.pentandra could be transmitted to tomatoplants. To examine whether G. pentandraplants may serve as a reservoir for thebegomoviruses during the off season, whitefly-mediated transmission assays from diseased G.pentandra to healthy tomato seedlings wereconducted. It was found that the presence ofbegomoviruses could be clearly detected 35and 42 days post-inoculation (Figs. 9, 10).Sequence analyses of the progeny ofbegomoviruses in tomato plants indicated thatthe TLCV-like sequences were selectivelyamplified in tomato plants, whereas only oneclone maintained the 3’-end sequences of theoriginal begomoviruses from G. pentandra (Fig.11). As the possibility of contamination wasruled out by the pre-inoculation screening withPCR, the observation suggested that a minor

population of TLCV-like begomoviruses in G.pentandra was selectively amplified whentransmitted to tomato plants by the whiteflies.The restriction of geminivirus host ranges hasalso previously been reported (Brown 1994;Brown et al. 1995). Our results confirm thatthe begomoviruses in G. pentandra canactually be transmitted by whiteflies to virus-free tomato plants, and indicate that hostfactors may play important roles in theselective amplification and recombination eventsof begomoviruses. Taken together, our observations suggestthat both weeds and B. tabaci play importantroles in the epidemiology of begomovirusdiseases, and should therefore be controlledmore rigorously in order to prevent severedamage caused by begomoviruses.

FUTURE STUDIES

The appearance of a new vector biotype andincreases in vector populations havecontributed to the emergence of geminivirus

Fig. 8. The phylogram depicting distance relationships of nucleotide sequences ofgeminiviruses coat protein from G. pentandra. The tlcv-twcp, Taiwanstrain of TLCV; ayvv-pdcp, Ping-Tung strain of AYVV; g9cp, gonocp, andgcp1 through gcp6, CP gene sequences of geminiviruses from G.pentandra. Scale bar represents 0.1 phylogenetic distance unit (0.1nucleotide substitution per position) (Huang et al. unpublished).

gcp4

gcp2

gcp3

g9cp

gcp1

gcp60.1

g1

gonocp

g3-2

tlcv-twcp

gcp5

ayw-pdcp

Fig. 9. (A) The presence of geminiviruses in the test tomato plants werescreened by PCR using primer GCP-1 and GCP-2 before inoculation.Lane M, 1 Kb marker; lane 1-5, tomato leaves; lane P, positive control(leaves from geminivirus-infected G. pentandra plant), (B) Leaves oftomato plants were analyzed at 28 dpi by PCR using primer GCP-1and GCP-2. Lane M, 1 Kb marker; lane 1-5, leaves from tomatoplants; lane P, positive control (Huang et al. unpublished).

Fig. 10. PCR detection of the presence of begomovirus DNAs in tomatoplants inoculated with viruliferous whiteflies. Leaves of tomato plantsinoculated by viruliferous whiteflies were analyzed at 42 dpi by PCRusing primer GCP-1 and GCP-2. Lane M, 1 Kb marker; lane 1-5,leaves from tomato plants; lane P, positive control. The position ofthe CP fragment is indicated on the right (Huang et al. unpublished).

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Fig. 11. Analyses of coat protein gene nucleotide sequences of the progeniesrecovered from whitefly transmission assays. The coat protein regionof the progeny geminiviruses were amplified by PCR, cloned andsequenced. The resulting nucleotide sequences were analyzed by theprogram PileUP. The alignment of the sequences, along with thereference sequences, is shown in (A). The phylogram depicting thegenetic relationships among the sequences is shown in (B). Scale barrepresents 0.1 phylogenetic distance unit (0.1 nucleotide substitutionper position) (Huang et al. unpublished).

icp1

icp6icp11

icp7

icp3icp2

icp10icp5

icp4tlcv-twcp

gcp5

icp9icp8

gonocpg9cp

disease problems. Thus, there is a need for abetter understanding of the factors that haveled to the increase in the vector populations indiverse cropping systems. The possibility thatvirus sequences are integrated into the hostgenome raises some major questions. Does themovement of host genotypes containing viralsequences lead to the emergence of newgeminivirus problems? Are these moleculesalso integrated into the host genome? Thecomplexities of emerging geminivirus problems

require a concerted effort by virologists as wellas entomologists and plant breeders to containfresh outbreaks and to minimize the damagecaused by geminivirus diseases to allowsustainable crop production (Varma andMalathi 2003). In the past decade, extensive and intensivesurveys of B. tabaci have been conducted todetect the spread of the B biotype, andidentify other biotypes within the B. tabacispecies complex. The diverse array of tools

15

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utilized for molecular analysis and interpretationof results has made it difficult to compareresults and draw conclusions. However,knowledge is advancing rapidly, and improvedunderstanding of B. tabaci systematics andepidemiologically useful distribution patterns arelikely in the near future. In consideration ofthe alarming rate with which new geminivirusesare emerging in tomato, there is an urgentneed to develop appropriate conceptualframeworks. It is probable that only throughthis multicomponent approach can economicallyand environmentally sound approaches bedeveloped for the successful management ofthese viruses (Oliveira et al. 2001). .

ACKNOWLEDGMENTS

We wish to express our sincere thanks to theFood and Fertilizer Technology Center (FFTC)for the Asian and Pacific Region for kindlyconducting this program. Thanks are likewiseextended to Dan Chamberlin for the Englishediting of the draft. Funding was provided inpart by the Bureau of Animal and Plant HealthInspection and Quarantine (Grant nos. 93AS-1.9.1-BQ-B2[4] and 94AS-13.3.1-BQ-B2[5]),Taipei, Taiwan ROC.

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